Epigenetic CRISPR Screening of 9p21.3 Non-Coding Regions identifies Cis-Regulatory Elements of P16 ^INK4a and P15 ^INK4b Controlling Cellular Senescence

Cellular senescence is a hallmark of aging and a promising target for extending human healthspan. Senescence is often accompanied by upregulation of the key senescence marker gene CDKN2A , yet the mechanism underlying its transcriptional activation remains unclear due to complex cis -regulations within the 9p21.3 locus. Here, we performed complementary CRISPR activation and interference screens in human mesenchymal stromal cells (MSCs) to systematically map non-coding cis -regulatory elements (CREs) at this locus that epigenetically regulate senescence. This approach revealed senescence-regulating CREs (SenReg-CREs) that bidirectionally modulate senescence through P16 ^INK4a and P15 ^INK4b . Notably, we identified a primate-specific short interspersed nuclear element (SINE) MIR3 embedded within the most potent distal SenReg-CRE. Deletion of this SINE:MIR3 accelerated senescence, revealing its potential insulator function in restraining CDKN2A/CDKN2B activation. Collectively, these findings reveal novel mechanisms underlying senescence-associated transcriptional activation of CDKN2A/CDKN2B and demonstrate that senescence is malleable through manipulation of regulatory element activity, highlighting the potential of epigenetically targeting these SenReg-CREs for senomorphic interventions.